tbuckley/cabot-search huggingface dataset:
Abstract P1017: Post-GWAS Functional Analysis Of CBLN2 Locus Reveals The Role Of CUX1 In The Inflammatory Pathogenesis Of Pulmonary Arterial Hypertension
Authors: Wei Sun, Danli Jiang, Ting Wu, Gang Li, Stephen Y Chan
Journal: Circulation Research (2023)

Background: Pulmonary arterial hypertension (PAH) is a deadly pulmonary vascular disease highly relevant to genetic susceptibility and acquired pathogenic triggers, especially inflammation. Chr18q22.3 near cerebellin 2 (CBLN2) gene is the first genetic locus identified by Genome-wide Association Study (GWAS) that is significantly associated with PAH risk in population of European ancestry. This locus includes a group of closely linked non-coding intergenic single nucleotide polymorphisms (SNPs) downstream of CBLN2. Although CBLN2 is upregulated in PAH and causes pulmonary vascular dysfunction, it remains unknown how these non-coding SNPs remotely affect CBLN2 and in turn alter the genetic susceptibility of PAH. Methods and Results: We applied Hi-C analysis, combined with electrophoretic mobility shift assay, to identify rs11151770 as a functional SNP (fSNP) characterized with allele-imbalanced nuclear protein binding and chromatin interaction with CBLN2 promoter, among 19 haplotype SNPs in a linkage disequilibrium (LD r 2 &gt;0.8) in CBLN2 locus. As an intergenic non-coding SNP, rs11151770 is located 110 kbp downstream of CBLN2 promoter. Proteomic analysis revealed nuclear transcription factor CUX1 binds specifically to the risk allele A of rs11151770 but not to the non-risk allele C. Acting as the activator for the expression of target gene CBLN2, CUX1 knockdown prevented, but CUX1 overexpression induced, the pathophenotypes of human pulmonary arterial endothelial cells (PAECs) associated with PAH. CUX1-CBLN2 was upregulated by acquired inflammatory PAH trigger IL-1β and was increased in lung tissues extracted from PAH patients. SNP genotyping in PAH patients and non-PAH controls showed a 2.2-fold risk allele A enrichment only in patients with PAH associated with connective tissue disorders, supporting a pathogenic mechanism of an inflammation-sensitive pathway (CUX1-CBLN2) combined with a PAH susceptible genotype (risk allele A of rs11151770) for the manifestation of clinical PAH. Conclusion: Our post-GWAS functional genomics analysis revealed a CUX1-mediated inflammatory pathway and provided a mechanistic explanation for the GWAS-identified association between the genetic variants in CBLN2 locus and PAH pathogenesis.
Abstract P2103: Cardiac Fibroblast-specific Role Of PTBP1 In Maintaining Heart Function
Authors: Shea N Ricketts, Shufen Chen, Li Qian
Journal: Circulation Research (2023)

Cardiac fibroblasts (CFs) are the primary nonmyocyte in the heart that maintain cardiac homeostasis by regulating myocardial function and cardiac remodeling. CFs secrete an extracellular matrix to stabilize the myocardial wall while maintaining the heart’s mechanical integrity during development and pathogenesis. Developmental and pathogenic transitions are directed through CFs by transcriptional and post-transcriptional modifications, such as alternative splicing. Alternative splicing is a central element in post-transcriptional gene regulation and proteomic diversity. Impaired and aberrant splicing is known to cause developmental defects and drive cardiac pathogenesis. RNA binding proteins (RBPs), which function as alternative splicing factors, are upregulated in cardiovascular disease states. While nearly a third of all CF gene regulation changes during cardiac fibrosis are subject to transcriptional regulation via RBPs, the role of CF specific splicing RBPs in the adult heart and during cardiac fibrosis is unknown. Our group previously identified high expression of well-known RBP and splicing factor polypyrimidine tract-binding protein (PTBP1) in CFs when compared to cardiomyocytes in the adult heart. With single cell transcriptomics we found PTBP1 regulates CF identity and plasticity, suggesting an emergent role of PTBP1 within CF biology. We hypothesize PTBP1 is essential for maintaining cardiac fibroblast function in the adult heart. Preliminary in vitro data suggests partial Ptbp1 knockdown impacts mechanical function by decreasing overall CF migration. To further characterize PTBP1 in vivo , we have validated our novel mouse model to specifically delete PTBP1 from CFs in adult mice. Following PTBP1 deletion, we will utilize an established model of fibrosis by Angiotensin II osmotic minipumps to characterize the CF specific PTBP1 loss during fibrosis. We will evaluate CF biology with our in vitro and in vivo models to examine PTBP1’s physiological and mechanistic role in maintaining CF biology during cardiac homeostasis and cardiac fibrosis pathogenesis.
Abstract P1001: Comprehensive “Tool Box” For Delineating Adrenergic Receptor Function
Authors: Joel Elmquist, Alexander Caron, Steven Wyler, Syann Lee, Yun-Hee Choi, Caleb Lord, Chen Liu, Teppei Fujikawa
Journal: Circulation Research (2023)

Sympathetic regulation of the cardiovascular system is required to maintain cardiac output and vascular tone in response to varied physiological stressors. This includes the release norepinepherine and epinephrine. Nor/epinephrine acts on nine, evolutionarily conserved, adrenergic, G-protein coupled receptors. Three alpha 1 ( Adra1 ), 3 alpha 2 ( Adra2 ), and 3 beta ( Adrb ) receptors. Understanding the distribution and cell-specific function of each of these receptors may lead to new targeted therapies for cardiovascular disease. To delineate the relative roles of each of the 9 adrenergic receptors, we are taking a genetic approach to study these receptors in mice; specifically, by using the Cre-loxP system to target the varied receptors in a tissue-specific manner. Unfortunately, there is a dearth of such tools to study these receptors. We therefore are in the process of generating three sets of tools for each of the 9 adrenergic receptors using the CRISPR-Cas9 method. 1. Adrenergic receptor floxed mice. 2. Adrenergic receptor reactivation (transcriptional blocking) mice and 3. Adrenergic receptor Cre mice. Thus far, we have generated Adra1a , Adra1b , Adra1d , Adra2a , Adrb2 , and Adrb3 floxed mice by inserting loxP sites flanking at least one exon near the 5’ end of the gene. The floxed mice were crossed to a germline-Cre (CMV-Cre), generating mice with whole-body loss of function alleles. Complementing the conditional loss of function alleles, we are also generating reactivation alleles by inserting a loxP-flanked transcriptional blocking cassette (TB) in each of the 9 endogenous adrenergic receptor genes. These TB alleles will allow us to reactivate endogenous expression of each adrenergic receptor in a Cre-dependent manner in an otherwise global knockout background. This model will allow us to identify tissues which are sufficient for the function of each adrenergic receptor. Finally, the ires-Cre models will allow for targeting of each adrenergic receptor-expressing cells with genetic reporters, and physiological studies using chemo and/or optogenetics. These new tools will assist in identifying the site of action and physiological function of various receptor subtypes and provide mechanistic insights into adrenergic receptor signaling.
Abstract P3089: Selective Activation Of Cardiac Macrophages To Study Injury And Repair
Authors: Rajesh K Kasam, Kelly M Grimes, Anne Katrine Z Johansen, Ronald J Vagnozzi, Jeffery D Molkentin
Journal: Circulation Research (2023)

Objective: Pathophysiological inflammatory responses play an important role during ischemia-mediated heart injury and remodeling, where monocytes and macrophages appear to be critically involved. Cardiac macrophages are generally classified as a recruited, monocyte-derived subset that is Ccr2 + and a tissue resident subset that is typically Ccr2 - . Recent studies have shown that these two subsets have divergent functions with Ccr2 - macrophages being pro-regenerative while Ccr2 + macrophages are pro-inflammatory. Here we selectively manipulated the content and/or activation of these two macrophage subsets in the heart. Methods and Results: To study the function of Ccr2 + cardiac macrophages in the heart, we have overexpressed Ccl-2 (Mcp-1) ligand specifically in cardiomyocytes by using adeno-associated virus serotype-9 (AAV9) mediated gene transfer. Postnatal day 4 (p4) Ccr2 RFP/+ pups were injected with either AAV9-Ccl-2 or AAV9-control. At two months of age, we observed increased Ccl-2 transcripts and protein expression in the heart as measured by qRT-PCR and ELISA, respectively. By quantifying immune cells using flow cytometry, we observed increased infiltration of Ccr2 + macrophages (CD45 + CD11b + CD64 + ) in the hearts of AAV9-Ccl2 injected mice compared to controls. However, echocardiography analysis revealed no change in cardiac function in adult Ccl-2 overexpressed mice. Moreover, the recruitment of Ccr2 + macrophages into the myocardium did not cause fibroblast expansion or activation. Conclusion: Our results show that cardiomyocyte-specific overexpression of Ccl-2 induces profound infiltration of Ccr2 + macrophages into the myocardium but this did not alter cardiac function or cause fibrosis at two or six months of age.
Abstract P1026: Structural And Biochemical Effects Of Missense MyBP-C Mutations On The Myofilament In Human Hypertrophic Cardiomyopathy
Authors: Christopher M McAllister, Vivek Jani, Devin L Nissen, Weikang Ma, David A Kass, Thomas C Irving, Sharlene Day
Journal: Circulation Research (2023)

Significance: While the disease mechanisms of truncating MyBP-C variants in HCM have been well defined, the mechanisms by which missense MyBP-C variants cause HCM are largely unknown. Recent studies show that unlike truncating variants that push myosin into an active high energy state due to their overall reduction in MyBP-C, missense MyBP-C properly incorporates into the sarcomere without affecting protein abundance. The consequences of missense MyBP-C on myosin have not been explored and may have implications for the treatment of HCM patients with missense MyBP-C variants. Results: We performed small angle X-ray diffraction (XrD) on human HCM tissue from patients with missense MyBP-C, and healthy control hearts. When healthy tissue was moved from relaxing pCa 8 to activating pCa 5 the I 1,1 /I 1,0 equatorial intensity ratio increased (p&lt;0.001) indicating myosin is moving away from the backbone and adopting an active conformation. However, in missense hearts I 1,1 /I 1,0 is lower than healthy hearts at pCa 8 meaning more myosin is localized to the backbone. This is contrary to other HCM mutations which are proposed to push myosin to an active conformation under relaxing conditions. I 1,1 /I 1,0 does not increase at pCa 5 (p&gt;0.05) indicating that at activating Ca myosin remains in the inactive conformation suggesting the thick filament Ca response is impaired in missense hearts. Ongoing Experiments: We are planning to perform XrD on more samples and Mant-ATP assays to determine if the structural inactivation of myosin in missense tissue also results in myosin remaining in the low energy super relaxed state (SRX). We expect the structural state to correlate with the biochemical state meaning missense hearts will demonstrate an equivalent percentage of SRX myosin compared to control hearts. Conclusions: Our finding that the transition of myosin to a structurally active conformation does not occur in human MyBP-C missense hearts distinguishes these variants from other HCM causing mutations such as truncating MyBP-C and missense MYH7 which have been shown to shift myosin to the active disordered relaxed state. If Mant-ATP assays show myosin is maintaining SRX along with an inactive conformation this suggests MyBP-C missense mutations act through a different mechanism.
Abstract P1014: Renal Autocrine Npff, Via Its Local Receptors, Regulates Blood Pressure Through Its Interaction With The Renal Dopaminergic System
Authors: Bibhas Amatya, Van Villar, Laureano D Asico, Shaun Moore, Jacob Polzin, Ines Armando, Robin A Felder, Pedro A Jose, Hewang Lee
Journal: Circulation Research (2023)

Neuropeptide FF (NPFF), a hormone originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the role of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, is unknown. In this study, we found that NPFF ( NPFF , Npff ), NPFF-R1( NPFFR1 , Npffr1 ), and NPFF-R2( NPFFR2 , Npffr2 ) mRNAs and proteins were expressed in both mouse and human renal proximal tubules (RPTs), determined by RT-PCR, RNAScope, immunoblotting, and immunofluorescence microscopy. NPFF-R2, compared with NPFF-R1, was predominantly expressed in RPTs and RPT cells (RPTCs). In human and mouse RPTCs, NPFF decreased cAMP production in a concentration- and time-dependent manner, consistent with its receptors’ linkage to the inhibitory G protein, Gαi. The D 1 R, but not D 5 R, colocalized with NPFF-R2 in human RPTCs and human kidney tissue sections. The colocalization between D 1 R and NPFF-R2 was decreased by the treatment with NPFF (100 nM, 30 min) in mouse RPTCs. Consistent with these findings, D 1 R co-immunoprecipitated with NPFF-R2 in human RPTCs. Fenoldopam, a D1-like receptor agonist, increased the cellular cAMP production and intracellular Na + accumulation, which were attenuated by NPFF (100 nM, 30 min), indicating an antagonistic interaction between NPFF and D1-like receptors. Moreover, acute and chronic administration of NPFF (10 μg in 100 μL, 0.5 μL/hr) in C57Bl/6 mice increased their blood pressures (from 96.4±3 to 114±5 mmHg, n=4) that were blocked by RF9 (10 μg in 100 μL, 0.5 μL/hr), an antagonist of both NPFF-R1 and NPFF-R2. NPFF also decreased urinary sodium excretion (from 0.36±0.06 to 0.28±0.05 mEq/day, n=4). In summary, NPFF by its negative interaction with the renal dopaminergic system regulates sodium excretion and blood pressure.
Abstract P1127: Induced Pluripotent Stem Cell-derived Macrophages Modulate Human Engineered Cardiac Tissue Function
Authors: Roberta I Lock, Pamela L Graney, Naveed Tavakol, Trevor Nash, Eloy Sanchez, Gordana Vunjak-Novakovic
Journal: Circulation Research (2023)

In the native human myocardium, cardiac resident macrophages are a critical mediator for normal cardiac function, performing a variety of biological roles in electrical conduction, regeneration, and phagocytosis and efferocytosis. However, most models of human engineered cardiac tissues (hECT), which aim to recapitulate the native adult myocardium for disease modeling and drug screening applications, do not include a macrophage population. Thus, in this study we introduced an iPSC-derived macrophage (iM0) population into an all iPSC-derived tissue engineered model of the human myocardium in order to assess its impact on tissue phenotype and function. CD14 + macrophages were differentiated from iPSCs and added as 7% of the total cell composition (7% iM0, 69.75% iCM, 23.25% iCF) in the hECT model. The hECTs were then cultured for 4 weeks and subjected to a ramped electrical stimulation protocol for matured function. The resulting tissues showed that the presence of a CD68 + macrophage population was maintained over this extended period of culture and significantly impacted hECT function, increasing contractile force production and modulating contraction and relaxation velocity. Interrogation of calcium signaling via isoproterenol revealed that iM0 presence in the tissues may be causing these functional differences via modulation of the cAMP signaling pathway. These observations demonstrate that macrophages significantly contribute to cardiac function in iPSC-derived hECT models and emphasize the need to further explore their contributions not only in healthy hECT models, but also in the contexts of disease and injury.
Abstract P1115: Epicardial Signaling Regulates Myocardial Contractility Via Mirna
Authors: Canan G Nebigil
Journal: Circulation Research (2023)

Background: A congenital left ventricular aneurysm is a rare cardiac malformation that maycause heartfailure, ventricular wall rupture, ventricular arrythmia or sudden cardiac death. However, mechanisms of congenital left ventricular aneurysm are not known. Paracrinesignals from cardiomyocytes, such as a cardiokine, prokineticin-2, can activate EPDCsdifferentiation in adult heart to promote neovascularization. How the reciprocal interaction viaprokineticin and miRs from epicardium to cardiomyocytes affect heart growth and function,and how EMT-related events contributecardiomyocyte proliferation and function areunknown. MicroRNA (miR124) has an important role in the adult heart, however, its role incardiac development is unknown. Results: Here in this study, by deletion of a prokineticin-2 receptor PKR1 through epicardialcell linage tracing at the different developmental stages of mice embryos, we found that thesemice exhibited ventricular rupture and embryonic lethality. Indeed, miR-124 was significantlyupregulated in PKR1 -deficient epicardial cells both in vitro and in vivo , whereas ectopicexpression of these miRs in epicardial cells resulted in the reduced expression of SNAI2 andsubsequently led to impaired EMT and EMT-related events such impaired migration andincreased apoptosis. Wild type mice cardiomyocytes that were cultured in condition mediumderived from PKR1-deficient epicardial cells had a lower beating rate and contractility. Treatment of the cardiomyocytes with miR-124mimic exhibited the same phenotype, that wasrestored by miR-124 inhibitor, indicating involvement of mir124 in epicardium-cardiomyocyte communication and regulation of cardiomyocyte contractility. Conclusion: These finding reveals a key signaling pathway in which a cardiokine,prokineticin-2 promotes EMT to form EPDCs and their migration in an autocrine manner, andsupports myocardial growth and contractility through repressing miR124. Our study mayprovide novel insight in etiology of congenital defects and new strategies to promotemyocardial regeneration.
Abstract P3056: Cardiac Fibroblast GSK-3α Contributes To Chronic Inflammation In The Ischemic Heart
Authors: Prachi Umbarkar, Suma Ejantkar, Sulivette Ruiz Ramirez, Sultan Tousif, Angelica Toro Cora, Qinkun Zhang, Hind Lal
Journal: Circulation Research (2023)

Background: Myocardial Infarction (MI) is the leading cause of death worldwide. Glycogen Synthase Kinase-3 (GSK-3) has been identified as a promising therapeutic target to treat cardiovascular diseases. The GSK-3 family has 2 isoforms, α and β. Previously, our lab has identified cardiac fibroblast (FB) GSK-3β acts as a negative regulator of fibrosis in the ischemic heart. However, the role of FB-GSK-3α in MI-induced adverse cardiac remodeling is not defined. Methods and Results: To determine the role of FB-GSK-3α in MI-induced adverse cardiac remodeling, GSK-3α was deleted specifically in the activated fibroblast/myofibroblast using tamoxifen (TAM)-inducible Periostin promoter-driven Cre recombinase system. At 12 weeks of age, mice were fed with the TAM diet. After 1 week of TAM treatment, control (GSK-3α fl/fl Cre -/- ) and KO (GSK-3α fl/fl Cre +/- ) mice were subjected to permanent LAD ligation (MI) surgery. Serial echocardiographic analysis revealed that FB-GSK-3α KO mice were protected from MI-induced systolic dysfunction and dilative remodeling. To investigate the role of FB-GSK-3α in MI-induced chronic inflammation, hearts were harvested at 4 weeks post-MI, and expression of signature inflammatory genes (TNFα, IL-6, IL-1β) was analyzed by qPCR. Surprisingly, the KO heart displayed remarkably low levels of inflammatory gene expression. To delineate underlying mechanisms, the effect of FB-GSK-3α deletion on the pro-inflammatory Nuclear factor kappa B (NF-κB) pathway was examined in vitro . Western blot analysis confirmed a significant reduction in NF-κB activation in GSK-3α-KO FBs. Importantly, the co-culture experiment demonstrated that the expression of inflammatory genes was downregulated in immune cells when cultured with GSK-3α KO FBs. These results indicate that GSK-3α can modulate immune cell phenotype through FB-immune cell crosstalk. Conclusion: Our findings suggest a critical role of FB-GSK-3α in pathological cardiac remodeling and heart failure that could be therapeutically targeted for future clinical applications.
Abstract P3162: Evaluating Cargo And Cardioprotective Effects Of Extracellular Vesicles From Human Right Atrial Appendage Mesenchymal Cells
Authors: Andreas Czosseck, Max M Chen, Chuan-Chih Hsu, Annette Meeson, Rachel Oldershaw, David Lundy
Journal: Circulation Research (2023)

Our team has previously shown the isolation and culture of a mesenchymal cell population from the right atrial appendage (RAA) of living human donor tissues. The cells are CD44/105/166 + , CD45/19/11b - and secrete growth factors (including VEGF and IGF-1) comparable to BM-MSCs. Their secretome preserved EF% and dP/dTmax in a mouse MI model and stimulated angiogenesis in a mouse limb ischemia model. In the current study, we specifically investigated using these cell-derived extracellular vesicles (EV) as a therapeutic. First, we validated our isolation technique and profiled EV miRNA cargo using PCR arrays. We then assessed the ability of EVs to protect human cardiomyocytes in a hypoxic (1% O2) hiPSC-CM model. Lastly, we used RNA-seq to compare the transcriptome of EV-treated hiPSC-CMs against hypoxic and normoxic controls. Results confirmed isolation of high purity EVs, showing (by cryoEM) ~120 nm bilayer membrane-bound vesicles, positive for CD81, CD63, ALIX, TSG101 and other EV markers (by antibody array). In the EV cargo we identified 363 known miRNAs present in 3 donor cell isolates, including hsa-miR-21-5p, -202-5p, -1260a and -125b. Target prediction of the most abundant miRNAs ranked GO:0010667 “negative regulation of cardiac muscle cell apoptotic process” as highly significant (26.3%, P = 1.6 x10 -8 ), as well as other predicted pathways related to angiogenesis, cardiac development and regulation of inflammation. A single, transient, treatment of donor EVs (66 ng EV protein/μl) reduced LDH leakage from hypoxic hiPSC-CMs, preserved morphology, and increased viability at 48 hrs and 7 days after injury. RNA-seq of the EV-treated hiPSC-CMs revealed strong upregulation of pathways related to PI3k-akt, calcium signaling and ECM binding compared to hypoxic hiPSC-CMs with vehicle control. We are currently validating key RNA-seq results. In conclusion, we analyzed and confirmed cardioprotective properties of EVs derived from human RAA samples using a human model system. Combined with our published animal studies, these data indicate that this cell population is a clinically-relevant source of donor cells for cardiac cell therapy. The EVs are also an attractive cell-free potential therapeutic option.